Simple method for sub-diffraction resolution imaging of cellular structures on standard confocal microscopes by three-photon absorption of quantum dots

This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres) and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts.

An improved algorithm for generating global window brightness temperatures from multiple satellite infrared imagery

An improved algorithm for the generation of gridded window brightness temperatures is presented. The primary data source is the International Satellite Cloud Climatology Project, level B3 data, covering the period from July 1983 to the present. The algorithm rakes window brightness, temperatures from multiple satellites, both geostationary and polar orbiting, which have already been navigated and normalized radiometrically to the National Oceanic and Atmospheric Administration's Advanced Very High Resolution Radiometer, and generates 3-hourly global images on a 0.5 degrees by 0.5 degrees latitude-longitude grid. The gridding uses a hierarchical scheme based on spherical kernel estimators. As part of the gridding procedure, the geostationary data are corrected for limb effects using a simple empirical correction to the radiances, from which the corrected temperatures are computed. This is in addition to the application of satellite zenith angle weighting to downweight limb pixels in preference to nearer-nadir pixels. The polar orbiter data are windowed on the target time with temporal weighting to account for the noncontemporaneous nature of the data. Large regions of missing data are interpolated from adjacent processed images using a form of motion compensated interpolation based on the estimation of motion vectors using an hierarchical block matching scheme. Examples are shown of the various stages in the process. Also shown are examples of the usefulness of this type of data in GCM validation.

Platelet 12-lipoxygenase activation via glycoprotein VI: involvement of multiple signaling pathways in agonist control of H(P)ETE synthesis

Lipoxygenases (LOX) contribute to vascular disease and inflammation through generation of bioactive lipids, including 12-hydro(pero xyeicosatetraenoic acid (12-H(P)ETE). The physiological mechanisms that acutely control LOX product generation in mammalian cells are uncharacterized. Human platelets that contain a 12-LOX isoform (p12-LOX) were used to define pathways that activate H(P)ETE synthesis in the vasculature. Collagen and collagen-related peptide (CRP) (1 to 10 g/mL) acutely induced platelet 12-H(P)ETE synthesis. This implicated the collagen receptor glycoprotein VI (GPVI), which signals via the immunoreceptor-based activatory motif (ITAM)- containing FcR chain. Conversely, thrombin only activated at high concentrations ( 0.2 U/mL), whereas U46619 and ADP alone were ineffective. Collagen or CRP-stimulated 12-H(P)ETE generation was inhibited by staurosporine, PP2, wortmannin, BAPTA/AM, EGTA, and L-655238, implicating src-tyrosine kinases, PI3-kinase, Ca2 mobilization, and p12-LOX translocation. In contrast, protein kinase C (PKC) inhibition potentiated 12-H(P)ETE generation. Finally, activation of the immunoreceptor tyrosine-based inhibitory motif (ITIM)– containing platelet endothelial cell adhesion molecule (PECAM-1) inhibited p12-LOX product generation. This study characterizes a receptor-dependent pathway for 12-H(P)ETE synthesis via the collagen receptor GPVI, which is negatively regulated by PECAM-1 and PKC, and demonstrates a novel link between immune receptor signaling and lipid mediator generation in the vasculature. (Circ Res. 2004;94:1598-1605.)

Specificity grafting of human antibody frameworks selected from a phage display library: generation of a highly stable humanized anti-CD22 single-chain Fv fragment

A prerequisite for the enrichment of antibodies screened from phage display libraries is their stable expression on a phage during multiple selection rounds. Thus, if stringent panning procedures are employed, selection is simultaneously driven by antigen affinity, stability and solubility. To take advantage of robust pre-selected scaffolds of such molecules, we grafted single-chain Fv (scFv) antibodies, previously isolated from a human phage display library after multiple rounds of in vitro panning on tumor cells, with the specificity of the clinically established murine monoclonal anti-CD22 antibody RFB4. We show that a panel of grafted scFvs retained the specificity of the murine monoclonal antibody, bound to the target antigen with high affinity (6.4-9.6 nM), and exhibited exceptional biophysical stability with retention of 89-93% of the initial binding activity after 6 days of incubation in human serum at 37degreesC. Selection of stable human scaffolds with high sequence identity to both the human germline and the rodent frameworks required only a small number of murine residues to be retained within the human frameworks in order to maintain the structural integrity of the antigen binding site. We expect this approach may be applicable for the rapid generation of highly stable humanized antibodies with low immunogenic potential.

Binaural prediction of speech intelligibility in reverberant rooms with multiple noise sources

When speech is in competition with interfering sources in rooms, monaural indicators of intelligibility fail to take account of the listener’s abilities to separate target speech from interfering sounds using the binaural system. In order to incorporate these segregation abilities and their susceptibility to reverberation, Lavandier and Culling [J. Acoust. Soc. Am. 127, 387–399 (2010)] proposed a model which combines effects of better-ear listening and binaural unmasking. A computationally efficient version of this model is evaluated here under more realistic conditions that include head shadow, multiple stationary noise sources, and real-room acoustics. Three experiments are presented in which speech reception thresholds were measured in the presence of one to three interferers using real-room listening over headphones, simulated by convolving anechoic stimuli with binaural room impulse-responses measured with dummy-head transducers in five rooms. Without fitting any parameter of the model, there was close correspondence between measured and predicted differences in threshold across all tested conditions. The model’s components of better-ear listening and binaural unmasking were validated both in isolation and in combination. The computational efficiency of this prediction method allows the generation of complex “intelligibility maps” from room designs. © 2012 Acoustical Society of America

My generation(s): cycles, branding and renewal in E4’s 'Skins'

In an era of fragmenting audience and diversified viewing platforms, youth television needs to move fast and make a lot of noise in order to capture and maintain the attention of the teenage viewer. British ensemble youth drama Skins (E4, 2007-2013) calls attention to itself with its high doses of drugs, chaotic parties and casual attitudes towards sexuality. It also moves quickly, shedding its cast every two seasons as they graduate from school, then renewing itself with a fresh generation of 16 year old characters - three cycles in total. This essay will explore the challenges of maintaining audience connections whilst resetting the narrative clock with each cycle. I suggest that the development of the Skins brand was key to the programme’s success. Branding is particularly important for an audience demographic who increasingly consume their television outside of broadcast flow and essential for a programme which renews its cast every two years. The Skins brand operate as a framework, as the central audience draw, have the strength to maintain audience connections when it ‘graduates’ those characters they identify with at the close of each cycle and starts again from scratch. This essay will explore how the Skins brand constructs a cohesive identity across its multiple generations, yet also consider how the cyclic form poses challenges for the programme’s representations and narratives. This cyclic form allows Skins to repeatedly reach out to a new audience who comes of age alongside each new generation and to reflect shifts in British youth culture. Thus Skins remains ever-youthful, seeking to maintain an at times painfully hip identity. Yet the programme has a somewhat schizophrenic identity, torn between its roots in British realist drama and surrealist comedy and an escapist aspirational glamour that shows the influence of US Teen TV. This combination results in a tendency towards a heightened melodrama at odds with Skins claims for authenticity - its much vaunted teenage advisors and young writers - with the cyclic structure serving to amplify the programme’s excessive tendencies. Each cycle wrestles with a need for continuity and familiarity - partly maintained through brand, aesthetic and setting - yet a desire for freshness and originality, to assert difference from what has gone before. I suggest that the inevitable need for each cycle to ‘top’ what has gone before results in a move away from character-based intimacy and the everyday to high-stakes drama and violence which sits uncomfortably within British youth television.